Tesis sobre el tema "Fusarium wilt of banana"

Genetic variation within the fungus F. oxysporum f.sp. cubense, which causes Fusarium wilt in banana, was examined using RAPD-PCR. RAPDPCR is a method of generating genome specific "fingerprints" which can be compared to determine genetic relatedness. In this study, the RAPD-PCR technique was optimised and used to determine the genetic relatedness between different vegetative compatibility groups (VCGs) and races, and among isolates within different VCGs and races of F. oxysporum f.sp. cubense. An understanding of the genetic diversity within F. oxysporum f.sp. cubense is essential to select or breed banana cultivars with durable resistance to Fusarium wilt; potentially resistant cultivars ideally should have resistance to all variants of the pathogen. Investigating genetic variation within the pathogen may also provide insight into the evolution of different populations and pathotypes of F. oxysporum f.sp. cubense.

Bananas (Musa sp.) serve as a staple diet and source of income for millions of people worldwide. The crop, however, is vulnerable to several important diseases such as Fusarium wilt, caused by the soilborne fungus Fusarium oxysporum f.sp. cubense (Foc) . During the mid-20th century, thousands of acres of export banana plantations were lost in Central America due to Fusarium wilt. The epidemic was brought under control only by replacing the highly susceptible Gros Michel banana with Cavendish cultivars. Cavendish bananas, however, are susceptible to Foe race 4, a highly virulent variant of the pathogen that also causes disease to banana varieties susceptible to Foc races 1 and 2. Only this time, no resistant replacement varieties acceptable to the commercial market exist, while cultural and/or chemical control strategies proved to be ineffective for disease control. The only sustainable solution, thus, would be the improvement of existing banana varieties that are acceptable to consumers for resistance to Fusarium wilt. An understanding of how plants defend themselves against pathogens is an imperative first step towards the development of disease-resistant plants. Two broad defence mechanisms against pathogen attack exist in plants: Constitutive defence mechanisms that are pathogen non-specific and induced plant defence responses following recognition of specific pathogen features. A primary objective of this study was to elucidate induced banana defence responses. Induced defence responses may include the induction of regulator and antifungal proteins, the production of active-oxygen species or products from the phenylpropanoid pathway. A few studies have attempted to elucidate the genetic factors involved in the banana defence response but to date no clear answer has arisen. Forward and reverse genetics encompass approached by which plant defence responses can be studied; a particular technique of interest which can be classified under both forward and reverse genetics is cDNA-Amplified Fragment Length Polymorphism (AFLP) In a field trial conducted in an Foc-infested field in Kiepersol, South Africa, cultivars Rose and Calcutta IV proved resistant, and FHIA-17 tolerant to Foe 'subtropical' race 4 (VCG 0120) when compared to the highly susceptible Cavendish cultivar 'Williams'. The genetic basis of defence to Foc in FHIA-17, Rose and Calcutta IV was verified using quantitative real-time reverse transcriptase PCR (qRT-RT-PCR). Catalase, POX and PAE were strongly up-regulated in the tolerant and resistant banana varieties. These genes are involved in the oxidative burst and secondary metabolism leading to the phenylpropanoid pathway and cell wall strengthening. Resistance to Foc, 'subtropical' race 4, thus seems to depend on the early recognition of the pathogen and subsequent blocking of its progress into the plant's vascular system. The response of resistant and tolerant banana varieties to Foc was elucidated by analysing the banana transcriptome 6 and 72 hours post inoculation (hpi) using cDNA-AFLP. Seventy-six differentially expressed transcript derived fragments (TDFs) were isolated, sequenced and subjected to BLASTX and BLASTN searches. Many of the sequences were not significantly similar to any other sequences in the databases, but several genes fragments showed homology to defence-related genes. TDFs representing genes such as S-adenosylmethionine synthase (SAMS) and isoflavone reductase, which are potentially involved in the production of cell wall strengthening compounds such as lignin, were identified. Expression patterns of selected TDFs as seen on the cDNA-AFLP gels were confirmed using qRT -PCR. As additional endogenous controls, two TDFs which displayed constitutive expression on the cDNA-AFLP gels, were isolated, sequenced and were optimised for use as endogenous control genes for the normalisation of the qRT-PCR data. In most cases, the expression patterns seen on the cDNA-AFLP gels were replicated by qRT-PCR. This study concludes that the tolerant (FHIA-17) and resistant (Rose and Calcutta IV) banana varieties induced defence-related genes upon attack by Foc and that the cDNA-AFLP technique was further effective in identifying additional defence-related genes. Looking to the future, the greatest understanding of the defence responses induced during the banana/Foe interaction would be obtained by using additional molecular approaches or techniques. It is in some cases sufficient to look exclusively at transcriptomic i.e. cDNA-AFLP and microarray data to study the plant's response, however, a look at differential protein and metabolite expression would complement transcriptomic data and add insight into the fate of certain expressed genes. This would possibly speed up the identification of the defence pathways used by resistant bananas to resist infection by Foc, once identified these pathways can be manipulated in the susceptible plants and thus the generation of a Foc resistant banana could finally become a reality.
Dissertation (MSc)--University of Pretoria, 2011.
Microbiology and Plant Pathology
unrestricted

Banana is one of the world’s most popular fruit crops and Sukali Ndizi is the most popular dessert banana in the East African region. Like other banana cultivars, Sukali Ndizi is threatened by several constraints, of which the Fusarium wilt disease is the most destructive. Fusarium wilt is caused by a soil-borne fungus, Fusarium oxysporum f.sp. cubense (Foc). No effective control strategy currently exists for this disease and although disease resistance exists in some banana cultivars, introducing resistance into commercial cultivars by conventional breeding is difficult because of low fertility. Considering that conventional breeding generates hybrids with additional undesirable traits, transformation is the most suitable way of introducing resistance in the banana genome. The success of this strategy depends on the availability of genes for genetic transformation. Recently, a novel strategy involving the expression of anti-apoptosis genes in plants was shown to result in resistance against several necrotrophic fungi, including Foc race 1 in banana cultivar Lady Finger. This thesis explores the potential of a plant-codon optimised nematode anti-apoptosis gene (Mced9) to provide resistance against Foc race 1 in dessert banana cultivar Sukali Ndizi. Agrobacterium-mediated transformation was used to transform embryogenic cell suspension of Sukali Ndizi with plant expression vector pYC11, harbouring maize ubiquitin promoter driven Mced9 gene and nptII as a plant selection marker. A total of 42 independently transformed lines were regenerated and characterized. The transgenic lines were multiplied, infected and evaluated for resistance to Foc race 1 in a small pot bioassay. The pathogenicity of the Ugandan Foc race 1 isolate used for infection was pre-determined and the spore concentration was standardised for consistent infection and symptom development. This process involved challenging tissue culture plants of Sukali Ndizi, a Foc race 1 susceptible cultivar and Nakinyika, an East African Highland cultivar known to be resistant to Foc race 1, with Fusarium inoculum and observing external and internal disease symptom development. Rhizome discolouration symptoms were the best indicators of Fusarium wilt with yellowing being an early sign of disease. Three transgenic lines were found to show significantly less disease severities compared to the wild-type control plants after 13 weeks of infection, indicating that Mced9 has the potential to provide tolerance to Fusarium wilt in Sukali Ndizi.

The banana industry worldwide is under threat from a fungal disease known as Fusarium wilt, a disease for which there is no chemical control. Conventional breeding approaches to generate resistant banana varieties are lengthy and very difficult. As such, genetic engineering for disease resistance is considered the most viable control option. In this PhD thesis, genetically modified banana plants were generated using several different stress tolerance genes. When challenged with Fusarium wilt in glasshouse trials, some lines showed increased resistance to the disease. The promising elite lines generated in this study will now require testing in field trials.

Bananas are susceptible to a diverse range of biotic and abiotic stresses, many of which cause serious production constraints worldwide. One of the most destructive banana diseases is Fusarium wilt caused by the soil-borne fungus, Fusarium oxysporum f. sp. cubense (Foc). No effective control strategy currently exists for this disease which threatens global banana production. Although disease resistance exists in some wild bananas, attempts to introduce resistance into commercially acceptable bananas by conventional breeding have been hampered by low fertility, long generation times and association of poor agronomical traits with resistance genes. With the advent of reliable banana transformation protocols, molecular breeding is now regarded as a viable alternative strategy to generate disease-resistant banana plants. Recently, a novel strategy involving the expression of anti-apoptosis genes in plants was shown to result in resistance against several necrotrophic fungi. Further, the transgenic plants showed increased resistance to a range of abiotic stresses. In this thesis, the use of anti-apoptosis genes to generate transgenic banana plants with resistance to Fusarium wilt was investigated. Since water stress is an important abiotic constraint to banana production, the resistance of the transgenic plants to water stress was also examined. Embryogenic cell suspensions (ECS) of two commercially important banana cultivars, Grand Naine (GN) and Lady Finger (LF), were transformed using Agrobacterium with the anti-apoptosis genes, Bcl-xL, Bcl-xL G138A, Ced-9 and Bcl- 2 3’ UTR. An interesting, and potentially important, outcome was that the use of anti-apoptosis genes resulted in up to a 50-fold increase in Agrobacterium-mediated transformation efficiency of both LF and GN cells over vector controls. Regenerated plants were subjected to a complete molecular characterisation in order to detect the presence of the transgene (PCR), transcript (RT-PCR) and gene product (Western blot) and to determine the gene copy number (Southern blot). A total of 36 independently-transformed GN lines (8 x Bcl-xL, 5 x Bcl-xL G138A, 15 x Ced-9 and 8 x Bcl-2 3’ UTR) and 41 independently-transformed LF lines (8 x Bcl-xL, 7 x BclxL G138A, 13 x Ced-9 and 13 x Bcl-2 3’ UTR) were identified. The 41 transgenic LF lines were multiplied and clones from each line were acclimatised and grown under glasshouse conditions for 8 weeks to allow monitoring for phenotypic abnormalities. Plants derived from 3 x Bcl-xL, 2 x Ced-9 and 5 x Bcl-2 3’ UTR lines displayed a variety of aberrant phenotypes. However, all but one of these abnormalities were off-types commonly observed in tissue-cultured, non-transgenic banana plants and were therefore unlikely to be transgene-related. Prior to determining the resistance of the transgenic plants to Foc race 1, the apoptotic effects of the fungus on both wild-type and Bcl-2 3’ UTR-transgenic LF banana cells were investigated using rapid in vitro root assays. The results from these assays showed that apoptotic-like cell death was elicited in wild-type banana root cells as early as 6 hours post-exposure to fungal spores. In contrast, these effects were attenuated in the root cells of Bcl-2 3’ UTR-transgenic lines that were exposed to fungal spores. Thirty eight of the 41 transgenic LF lines were subsequently assessed for resistance to Foc race 1 in small-plant glasshouse bioassays. To overcome inconsistencies in rating the internal (vascular discolouration) disease symptoms, a MatLab-based computer program was developed to accurately and reliably assess the level of vascular discolouration in banana corms. Of the transgenic LF banana lines challenged with Foc race 1, 2 x Bcl-xL, 3 x Ced-9, 2 x Bcl-2 3’ UTR and 1 x Bcl-xL G138A-transgenic line were found to show significantly less external and internal symptoms than wild-type LF banana plants used as susceptible controls at 12 weeks post-inoculation. Of these lines, Bcl-2 3’ UTR-transgenic line #6 appeared most resistant, displaying very mild symptoms similar to the wild-type Cavendish banana plants that were included as resistant controls. This line remained resistant for up to 23 weeks post-inoculation. Since anti-apoptosis genes have been shown to confer resistance to various abiotic stresses in other crops, the ability of these genes to confer resistance against water stress in banana was also investigated. Clonal plants derived from each of the 38 transgenic LF banana plants were subjected to water stress for a total of 32 days. Several different lines of transgenic plants transformed with either Bcl-xL, Bcl-xL G138A, Ced-9 or Bcl-2 3’ UTR showed a delay in visual water stress symptoms compared with the wild-type control plants. These plants all began producing new growth from the pseudostem following daily rewatering for one month. In an attempt to determine whether the protective effect of anti-apoptosis genes in transgenic banana plants was linked with reactive oxygen species (ROS)-associated programmed cell death (PCD), the effect of the chloroplast-targeting, ROS-inducing herbicide, Paraquat, on wild-type and transgenic LF was investigated. When leaf discs from wild-type LF banana plants were exposed to 10 ìM Paraquat, complete decolourisation occurred after 48 hours which was confirmed to be associated with cell death and ROS production by trypan blue and 3,3-diaminobenzidine (DAB) staining, respectively. When leaf discs from the transgenic lines were exposed to Paraquat, those derived from some lines showed a delay in decolourisation, suggesting only a weak protective effect from the transgenes. Finally, the protective effect of anti-apoptosis genes against juglone, a ROS-inducing phytotoxin produced by the causal agent of black Sigatoka, Mycosphaerella fijiensis, was investigated. When leaf discs from wild-type LF banana plants were exposed to 25 ppm juglone, complete decolourisation occurred after 48 hours which was again confirmed to be associated with cell death and ROS production by trypan blue and DAB staining, respectively. Further, TdT-mediated dUTP nick-end labelling (TUNEL) assays on these discs suggested that the cell death was apoptotic. When leaf discs from the transgenic lines were exposed to juglone, discs from some lines showed a clear delay in decolourisation, suggesting a protective effect. Whether these plants are resistant to black Sigatoka is unknown and will require future glasshouse and field trials. The work presented in this thesis provides the first report of the use of anti-apoptosis genes as a strategy to confer resistance to Fusarium wilt and water stress in a nongraminaceous monocot, banana. Such a strategy may be exploited to generate resistance to necrotrophic pathogens and abiotic stresses in other economically important crop plants.

Bananas are susceptible to a diverse range of biotic and abiotic stresses, many of which cause serious production constraints worldwide. One of the most destructive banana diseases is Fusarium wilt caused by the soil-borne fungus, Fusarium oxysporum f. sp. cubense (Foc). No effective control strategy currently exists for this disease which threatens global banana production. Although disease resistance exists in some wild bananas, attempts to introduce resistance into commercially acceptable bananas by conventional breeding have been hampered by low fertility, long generation times and association of poor agronomical traits with resistance genes. With the advent of reliable banana transformation protocols, molecular breeding is now regarded as a viable alternative strategy to generate disease-resistant banana plants. Recently, a novel strategy involving the expression of anti-apoptosis genes in plants was shown to result in resistance against several necrotrophic fungi. Further, the transgenic plants showed increased resistance to a range of abiotic stresses. In this thesis, the use of anti-apoptosis genes to generate transgenic banana plants with resistance to Fusarium wilt was investigated. Since water stress is an important abiotic constraint to banana production, the resistance of the transgenic plants to water stress was also examined. Embryogenic cell suspensions (ECS) of two commercially important banana cultivars, Grand Naine (GN) and Lady Finger (LF), were transformed using Agrobacterium with the anti-apoptosis genes, Bcl-xL, Bcl-xL G138A, Ced-9 and Bcl- 2 3’ UTR. An interesting, and potentially important, outcome was that the use of anti-apoptosis genes resulted in up to a 50-fold increase in Agrobacterium-mediated transformation efficiency of both LF and GN cells over vector controls. Regenerated plants were subjected to a complete molecular characterisation in order to detect the presence of the transgene (PCR), transcript (RT-PCR) and gene product (Western blot) and to determine the gene copy number (Southern blot). A total of 36 independently-transformed GN lines (8 x Bcl-xL, 5 x Bcl-xL G138A, 15 x Ced-9 and 8 x Bcl-2 3’ UTR) and 41 independently-transformed LF lines (8 x Bcl-xL, 7 x BclxL G138A, 13 x Ced-9 and 13 x Bcl-2 3’ UTR) were identified. The 41 transgenic LF lines were multiplied and clones from each line were acclimatised and grown under glasshouse conditions for 8 weeks to allow monitoring for phenotypic abnormalities. Plants derived from 3 x Bcl-xL, 2 x Ced-9 and 5 x Bcl-2 3’ UTR lines displayed a variety of aberrant phenotypes. However, all but one of these abnormalities were off-types commonly observed in tissue-cultured, non-transgenic banana plants and were therefore unlikely to be transgene-related. Prior to determining the resistance of the transgenic plants to Foc race 1, the apoptotic effects of the fungus on both wild-type and Bcl-2 3’ UTR-transgenic LF banana cells were investigated using rapid in vitro root assays. The results from these assays showed that apoptotic-like cell death was elicited in wild-type banana root cells as early as 6 hours post-exposure to fungal spores. In contrast, these effects were attenuated in the root cells of Bcl-2 3’ UTR-transgenic lines that were exposed to fungal spores. Thirty eight of the 41 transgenic LF lines were subsequently assessed for resistance to Foc race 1 in small-plant glasshouse bioassays. To overcome inconsistencies in rating the internal (vascular discolouration) disease symptoms, a MatLab-based computer program was developed to accurately and reliably assess the level of vascular discolouration in banana corms. Of the transgenic LF banana lines challenged with Foc race 1, 2 x Bcl-xL, 3 x Ced-9, 2 x Bcl-2 3’ UTR and 1 x Bcl-xL G138A-transgenic line were found to show significantly less external and internal symptoms than wild-type LF banana plants used as susceptible controls at 12 weeks post-inoculation. Of these lines, Bcl-2 3’ UTR-transgenic line #6 appeared most resistant, displaying very mild symptoms similar to the wild-type Cavendish banana plants that were included as resistant controls. This line remained resistant for up to 23 weeks post-inoculation. Since anti-apoptosis genes have been shown to confer resistance to various abiotic stresses in other crops, the ability of these genes to confer resistance against water stress in banana was also investigated. Clonal plants derived from each of the 38 transgenic LF banana plants were subjected to water stress for a total of 32 days. Several different lines of transgenic plants transformed with either Bcl-xL, Bcl-xL G138A, Ced-9 or Bcl-2 3’ UTR showed a delay in visual water stress symptoms compared with the wild-type control plants. These plants all began producing new growth from the pseudostem following daily rewatering for one month. In an attempt to determine whether the protective effect of anti-apoptosis genes in transgenic banana plants was linked with reactive oxygen species (ROS)-associated programmed cell death (PCD), the effect of the chloroplast-targeting, ROS-inducing herbicide, Paraquat, on wild-type and transgenic LF was investigated. When leaf discs from wild-type LF banana plants were exposed to 10 ìM Paraquat, complete decolourisation occurred after 48 hours which was confirmed to be associated with cell death and ROS production by trypan blue and 3,3-diaminobenzidine (DAB) staining, respectively. When leaf discs from the transgenic lines were exposed to Paraquat, those derived from some lines showed a delay in decolourisation, suggesting only a weak protective effect from the transgenes. Finally, the protective effect of anti-apoptosis genes against juglone, a ROS-inducing phytotoxin produced by the causal agent of black Sigatoka, Mycosphaerella fijiensis, was investigated. When leaf discs from wild-type LF banana plants were exposed to 25 ppm juglone, complete decolourisation occurred after 48 hours which was again confirmed to be associated with cell death and ROS production by trypan blue and DAB staining, respectively. Further, TdT-mediated dUTP nick-end labelling (TUNEL) assays on these discs suggested that the cell death was apoptotic. When leaf discs from the transgenic lines were exposed to juglone, discs from some lines showed a clear delay in decolourisation, suggesting a protective effect. Whether these plants are resistant to black Sigatoka is unknown and will require future glasshouse and field trials. The work presented in this thesis provides the first report of the use of anti-apoptosis genes as a strategy to confer resistance to Fusarium wilt and water stress in a nongraminaceous monocot, banana. Such a strategy may be exploited to generate resistance to necrotrophic pathogens and abiotic stresses in other economically important crop plants.

Banana is an important staple food crop however, production of the world’s most widely planted variety, the Cavendish banana, is threatened by a devastating fungal disease, called Fusarium wilt. Fusarium wilt of Cavendish bananas in the tropics is caused by Fusarium oxysporum f. sp. cubense (Foc) ‘tropical’ race 4 (TR4), while a variant of the fungus causing the disease in the subtropics is called Foc ‘subtropical’ race 4 (STR4). The incidence of Fusarium wilt in the subtropics is usually aggravated after winter, which suggests that the plant is predisposed to Foc STR4 during cold temperatures. The objective of this study was to investigate the molecular processes, in both Foc and banana, which contribute to Fusarium wilt development under cold stress. cDNA-AFLP expression profiling was used to elucidate the transcriptome of Foc STR4, Foc TR4 and non-pathogenic F. oxysporum isolates on minimal medium (without carbon source). This resulted in the identification of 229 unique gene fragments which included transcript derived fragments (TDFs) encoding for chitinase class V (chsV), GTPase activating protein, Major Facilitator Superfamily (MFS) multidrug transporter and serine/threonine protein kinase (ste12) genes. We speculate that those genes play a role in escaping host defence responses, and result in cell wall degradation. Pathogenicity-related genes identified in other formae speciales of F. oxysporum, such as the sucrose non-fermenting, F-box protein required for pathogenicity genes (frp1) and cyp55, were significantly up-regulated in Foc STR4 and Foc TR4, but not in F. oxysporum isolates non-pathogenic to banana. We suggest that these genes are important for the Fusarium wilt pathogen to enter the host xylem tissue, as they regulate the abundance of cell wall degrading enzymes. The increase in expression of cyp55 in pathogenic F. oxysporum may give the pathogen the ability to regulate the nitrogen response pathway, which is essential for pathogenicity. This study provided the first identification of genes in Foc that potentially contribute to pathogenicity in banana. Cavendish banana plants subjected to cold temperatures and inoculated with Foc resulted in a significant increase in disease severity. Visual symptoms, however, only appeared in inoculated plants after they were transferred to 28oC. Transcriptome analysis showed that several general defence mechanisms are activated in Cavendish bananas infected with Foc. An important finding was that expression of defencerelated genes was delayed in cold-treated plants, which enhance disease severity. More specifically, the induction of PR genes (PR-1, PR-4, PR-6 and PR-10), C4H, involved in phenylpropanoid pathway and thus important for phytoalexin and lignin production, and Hin1, involved in the hypersensitive response, was significantly suppressed at an early stage during cold stress. This potentially provides an opportunity to Foc STR4 to invade the xylem and progress within the vascular bundles before plant defences are activated. Disease development mainly occurs at 28oC, as the pathogen prefers higher temperatures for optimal growth and sporulation. At this temperature, movement of water through the vascular vessels of the roots and pseudostem is increased, resulting in the more rapid wilting of affected plants. Thus, cold stress may enhance infection of Foc STR4.
Thesis (PhD)--University of Pretoria, 2013.
gm2013
Microbiology and Plant Pathology
Unrestricted

A doença da bananeira \'mal-do-Panamá\', causada pelo fungo Fusarium oxysporum f. sp. cubense (Foc) é uma das doenças mais destrutivas da bananeira e é considerada uma das seis doenças economicamente mais importante da história da humanidade. Algumas cultivares resistentes, como a \'BRS Platina\', foram lançadas pela Embrapa, porém para a sustentabilidade da resistência é necessário entender os mecanismos moleculares envolvidos na resposta de resistência e defesa. O objetivo deste estudo foi caracterizar o processo de infecção pelo Foc raça 1 em três cultivares contrastantes para a resistência e analisar o padrão transcricional no início da interação. A análise histopatológica indicou que o Foc raça 1 penetra pela raízes laterais e principal, colonizando os espaços inter e intracelular do córtex nas três cultivares. Foram visualizadas, hifas \'globosas\' na cultivar suscetível \'Maçã\' com a formação de estruturas de resistência, como clamidósporos. Na cultivar resistente \'BRS Platina\', foi observado por microscopia óptica no período inicial da interação (24 horas após inoculação) a indução de respostas de defesa da planta, como formação de zona de cicatrização, e aos 15 dias após inoculação, formação de tilose, presença de cristais de oxalato de cálcio e deposição de calose. Foi utilizada a tecnologia Illumina para sequenciamento massal de RNA e abordagens de bioinformática para identificar genes diferencialmente expressos (DE) relacionados com a resposta de defesa de bananeira em interações compatíveis e incompatíveis. O sequenciamento paired-end gerou um total de 113.632.486 fragmentos (reads) com alta qualidade. Do total de reads alinhados no genoma referência (\'DH-Pahang\'), 55.555.480 alinharam-se com genes conhecidos e anotados no genoma referência, sendo utilizados para a análise DE inoculado x não inoculado, permitindo detectar 2.307 genes para as três cultivares. Os genes anotados de cada cultivar foram comparados, sendo identificados quatro genes comuns para as três cultivares, dez compartilhados entre \'Maçã\' e \'Prata-anã\', 21 compartilhados entre \'BRS Platina\' e \'Maçã\', 114 compartilhados entre \'BRS Platina\' e \'Prata-anã\', além de 75 serem exclusivos de \'Maçã\', 599 de \'BRS Platina\' e 1484 de \'Prata-anã\'. O mecanismo de resistência/defesa ao Foc em \'BRS Platina\', ocorre em nível de percepção precoce na presença do patógeno desencadeando resposta de defesa inexistente em \'Maçã\', e com cinética distinta da cultivar com resposta intermediária (\'Prata-anã\'). Dessa forma, os resultados permitiram propor um modelo da resposta de defesa/resistência ao Foc raça 1 em bananeira, baseando-se no nível de indução de genes que codificam para proteínas de reconhecimento do patógeno (receptor like kinase), fatores de transcrição (WRKY e MYB); reforço e síntese de parede celular, degradação da parede celular do fungo (quitinase e glucanases), heat shocks, enzimas antioxidantes e na resposta visualizada pela histologia na cultivar \'BRS Platina\'. Sendo assim, este trabalho fornece novas perspectivas para estudos de análise funcional, identificação e anotação de novos genes relacionados a resposta de defesa e resistência ao Foc raça 1.
The banana Panama disease, caused by fungus Fusarium oxysporum f. sp. cubense (Foc), is one of the most destructive disease of the industry, and it is considered one of the six most economically important of all times. A few cultivars, such as \'BRS Platina\', were released, but it is still necessary to understand molecular mechanisms involved in defense response and resistance. The objective of this study was to characterize the infection process by Foc in three banana cultivars contrasting for resistance to Foc and to analyze the transcriptional profile at the beginning of interaction. In this way, Foc race 1 penetrated the main and lateral roots, colonizing inter- and intracellular spaces of the root cortex in the three cultivars. Hyphae were globose in the susceptible cultivar \'Maçã\' with the formation of resilience structure, such as chlamydospores. In the resistant cultivar \'BRS Platina\', during the initial period of interaction (24 hours after inoculation), induced of plant defense responses, such as a healing zone, tylosis formation, presence of calcium oxalate and callose deposition. The Illumina technology were applied to sequence RNA, followed by bioinformatic tools to identify genes differentially expressed (DE) related to resistance and defense response in the compatible and incompatible interactions. Pair-end sequencing generated a total of 113,632,486 reads with high quality. From the total of aligned reads to the banana reference genome (\'DH-Pahang\'), 55,555,480 aligned with gene models annotated in the reference genome. The aligned contigs were analysed for DE, comparing inoculated x non-inoculated, enabling the detection of 2307 genes for the three cultivars. Each annotated gene from each cultivar was compared: four common genes to the three cultiars; 10 genes were shared between \'Maçã\' and \'Prata-anã\'; 21 shared between \'BRS Platina\' and \'Maçã\'; 114 shared between \'BRS Platina\' and \'Prata-anã\', plus 75 exclusive to \'Maçã\'; 599 exclusive to \'BRS Platina\' and 1,484 to \'Prata-anã\'. The mechanism of resistance/defense in \'BRS Platina\', level of perception occurs early in the presence of the pathogen defense response triggering nonexistent in \'Maçã\' and with kinetics distinct cultivar with intermediate response (\'Prata-anã\'). Thus, the results have provided a model of defense response/resistance to Foc race 1 in banana, based on the level of gene induction that encode recognition proteins (Receptor-like Kinase, RLK), transcription factors (WRKY and MYB), cell wall synthesis and reinforcement, degradation of fungal cell wall (chitinases and glucanases), heat shocks , proteins;anto-oxidative enzymes and visualized by histologcal in response cultivar \'BRS Platina\'. The present work offer new perspectives to functional analyses, identification and annotation of new genes related to resistance and defense response to Foc race 1.

Bananas (Musa sp) are one of the most important food crops in the world and provide a staple food and source of income in many households especially in Africa. Diseases are a major constraint to production with bunchy top, caused by Banana bunchy top virus (BBTV) generally considered the most important virus disease of bananas worldwide. Of the fungal diseases, Fusarium wilt, caused by the Fusarium oxysporum f.sp cubense (Foc), and black Sigatoka, caused by Mycosphaerella fijiensis, are arguably two of the most important and cause significant yield losses. The low fertility of commercially important banana cultivars has hampered efforts to generate disease resistance using conventional breeding. Possible alternative strategies to generate or increase disease resistance are through genetic engineering or by manipulation of the innate plant defence mechanisms, namely systemic acquired resistance (SAR). The first research component of this thesis describes attempts to generate BBTV-resistant banana plants using a genetic modification approach. The second research component of the thesis focused on the identification of a potential marker gene associated with SAR in banana plants and a comparison of the expression levels of the marker gene in response to biotic and abiotic stresses, and chemical inducers. Previous research at QUT CTCB showed that replication of BBTV DNA components in banana embryogenic cell suspensions (ECS) was abolished following co-bombardment with 1.1mers of mutated BBTV DNA-R. BBTV DNA-R encodes the master replication protein (Rep) and is the only viral protein essential for BBTV replication. In this study, ECS of banana were stably transformed with the same constructs, each containing a different mutation in BBTV DNA-R, namely H41G, Y79F and K187M, to examine the effect on virus replication in stably transformed plants. Cells were also transformed with a construct containing a native BBTV Rep. A total of 16, 16, 11 and five lines of stably transformed banana plants containing the Y79F, H41G, K187M and native Rep constructs, respectively, were generated. Of these, up to nine replicates from Y79F lines, four H41G lines, seven K187M lines and three native Rep lines were inoculated with BBTV by exposure to viruliferous aphids in two separate experiments. At least one replicate from each of the nine Y79F lines developed typical bunchy top symptoms and all tested positive for BBTV using PCR. Of the four H41G lines tested, at least one replicate from three of the lines showed symptoms of bunchy top and tested positive using PCR. However, none of the five replicates of one H41G line (H41G-3) developed symptoms of bunchy top and none of the plants tested positive for BBTV using PCR. Of the seven K187M lines, at least one replicate of all lines except one (K187M-1) developed symptoms of bunchy top and tested positive for BBTV. Importantly, none of the four replicates of line K187M-1 showed symptoms or tested positive for BBTV. At least one replicate from each of the three native Rep lines developed symptoms and tested positive for BBTV. The H41G-3 and K187M-1 lines possibly represent the first transgenic banana plants generated using a mutated Rep strategy. The second research component of this thesis focused on the identification of SAR-associated genes in banana and their expression levels in response to biotic and abiotic stresses and chemical inducers. The impetus for this research was the observation that tissue-cultured (TC) banana plants were more susceptible to Fusarium wilt disease (and possibly bunchy top disease) than plants grown from field-derived suckers, possibly due to decreased levels of SAR gene expression in the former. In this study, the pathogenesis-related protein 1 (PR-1) gene was identified as a potential marker for SAR gene expression in banana. A quantitative real-time PCR assay was developed and optimised in order to determine the expression of PR-1, with polyubiquitin (Ubi-1) found to be the most suitable reference gene to enable relative quantification. The levels of PR-1 expression were subsequently compared in Lady Finger and Cavendish (cv. Williams) banana plants grown under three different environmental conditions, namely in the field, the glass house and in tissue-culture. PR-1 was shown to be expressed in both cultivars growing under different conditions. While PR-1 expression was highest in the field grown bananas and lowest in the TC bananas in Lady Finger cultivar, this was not the case in the Cavendish cultivar with glass house plants exhibiting the lowest PR-1 expression compared with tissue culture and field grown plants. The important outcomes of this work were the establishment of a qPCR-based assay to monitor PR-1 expression levels in banana and a preliminary assessment of the baseline PR-1 expression levels in two banana cultivars under three different growing conditions. After establishing the baseline PR-1 expression levels in Cavendish bananas, a study was done to determine whether PR-1 levels could be increased in these plants by exposure to known banana pathogens and non-pathogens, and a known chemical inducer of SAR. Cavendish banana plants were exposed to pathogenic Foc subtropical race 4 (FocSR4) and non-pathogenic Foc race 1 (Foc1), as well as two putative inducers of resistance, Fusarium lycopersici (Fol) and the chemical, acibenzolar-S-methyl (BION®). Tissue culture bananas were acclimatised under either glass house (TCS) or field (TCH) conditions and treatments were carried out in a randomised complete block design. PR-1 expression was determined using qPCR for both TCS and TCH samples for the period 12-72h post-exposure. Treatment of TCH plants using Foc1 and FocSR4 resulted in 120 and 80 times higher PR-1 expression than baseline levels, respectively. For TCS plants treated with Foc1, PR-1 expression was 30 times higher than baseline levels at 12h post-exposure, while TCS plants treated with FocSR4 showed the highest PR-1 expression (20 times higher than baseline levels) at 72h post-exposure. Interestingly, when TCS plants were treated with Fol there was a marked increase of PR-1 expression at 12 h and 48 h following treatment which was 4 and 8 times higher than the levels observed when TCS plants were treated with Foc1 and FocSR4, respectively. In contrast, when TCH plants were treated with Fol only a slight increase in PR-1 expression was observed at 12 h, which eventually returned to baseline levels. Exposure of both TCS and TCH plants to BION® resulted in no effect on PR-1 expression levels at any time-point. The major outcome of the SAR study was that the glass house acclimatised tissue culture bananas exhibited lower PR-1 gene expression compared to field acclimatised tissue culture plants and the identification of Fol as a good candidate for SAR induction in banana plants exhibiting low PR-1 levels. A number of outcomes that foster understanding of both pathogen-derived and plant innate resistance strategies in order to potentially improve banana resistance to diseases were explored in this study and include identification of potential inducers of systemic acquired resistance and a promising mutated Rep approach for BBTV resistance. The work presented in this thesis is the first report on the generation of potential BBTV resistant bananas using the mutated Rep approach. In addition, this is the first report on the status of SAR in banana grown under different conditions of exposure to the biotic and abiotic environment. Further, a robust qPCR assay for the study of gene expression using banana leaf samples was developed and a potential inducer of SAR in tissue culture bananas identified which could be harnessed to increase resistance in tissue culture bananas.

The toxigenicity of isolates of Fusarium for chickpea, Cicer arietinum, the third most important legume crop in the world was studied. Fungi were grown in liquid culture and the culture filtrates assayed on cells isolated from leaflets of the plant. One isolate, designated FOC 5, produced cultures that were predominately red (70-80% of the cultures). When the culture filtrates of all isolates over time were assayed, the red cultures of FOC 5 were much more toxic than those of the other isolates and were also about 10 times more toxic than the colourless cultures of FOC 5. Toxic titres of the red FOC 5 cultures peaked at 12 days when grown at 20 C. The toxin from these red cultures were purified by solvent partitioning, solid phase extraction (SPE), thin layer chromatography (TLC) and high performance liquid chromatography (HPLC) using the assay to monitor the stages in purification. Shaking of culture filtrates of FOC 5 with ethyl acetate resulted in about half the toxic activity (50-55%) partitioning into the organic phase and 25-30% remaining in the aqueous phase. The activity of the aqueous phase was lost on freeze-drying suggesting a volatile compound. When the ethyl acetate phase was dissolved in aqueous acetonitrile and applied to C18 SPE cartridges, about 9% was not adsorbed and 35% could be eluted with methanol. Greater affinity was shown for cyano SPE cartridges with 6% not adsorbed and 45 % recoverable by elution in acetonitrile. Attempts at purification of the toxin(s) of adsorbed and non-adsorbed fractions from these reversed phase cartridges by HPLC did not yield pure products. Recovery of activity of the ethyl acetate phase from flash chromatography on silica gel was 61-110%. However, HPLC demonstrated that several compounds were present in the active fractions Separation of components of the ethyl acetate phase or the fraction adsorbed by cyano cartridges of culture filtrates by TLC on silica gel rather than using SPE, flash or reversed phase HPLC was more successful. Red bands corresponding to the active compound were scraped from TLC plates and eluted in acetonitrile. HPLC of the eluents on a cyano column with 10% acetonitrile as the mobile phase demonstrated a single homogeneous peak with absorption maxima of 224 and 281 nm. The purified fraction is, at the time of writing, being studied by Professor Mike Beale at Rothamsted Research using nuclear magnetic resonance techniques in order to determine its structure. Four other isolates, identified by the International Crops Research Institute for the Semi-Arid Tropics as F. oxysporum f. sp. ciceri did not produce the red, toxic compound, throwing doubt on the correct identification of the isolates. When the sequences of ribosomal DNA of all five isolates were determined, the isolate that produced the red toxic compound most closely matched Fusarium acutatum (99%), in a BLAST search and this accorded with its morphology. A BLAST search showed that three of the other isolates matched the sequence of cotton pathogen, F. oxysporum f. sp. vasinfectum (100%, 100% and 97%) and one closely matched F. oxysporum f. sp. vanillae (99%) These results suggest that a re-evaluation of the taxonomy of Fusarium species causing wilt of chickpea is required.

3 pp.
This publication provides information on the development and management of Fusarium wilt of lettuce. Topics covered include the characteristics of the plant pathogen, disease development, and disease management considerations.

In the 2001-2002 production season, Fusarium wilt was observed for the first time in six different lettuce fields in the Gila and Dome Valley production areas of Yuma County, Arizona. The disease was found in 11 additional sites during 2002-2003. Fusarium wilt presents a serious threat to the health of the lettuce industry in Arizona. The only effective means of controlling Fusarium wilt of lettuce at this time is to avoid infested fields. On the other hand, Fusarium wilt in other crops, such as tomatoes and melons, is controlled effectively by planting cultivars resistant to the pathogen. The relative resistance of lettuce cultivars grown in the Arizona desert production region is unknown; therefore, a cultivar evaluation trial was established in a field known to contain the wilt pathogen, Fusarium oxysporum f.sp. lactucae. Tested cultivars were grouped into three different planting dates: Sep 7, Oct 17 and Dec 6, 2002. A majority of the cultivars within each planting date were those that would be planted in the desert at that time. Fusarium wilt was severe in the early planting of lettuce (Sep 7), moderate in the second planting (Oct 17) and very mild in the third planting (Dec 6). Disease severity was low in some lettuce cultivars in the second planting and most cultivars in the third planting. Among the types of lettuce tested, head lettuce was usually least resistant whereas romaine was most resistant. The data presented in this report are preliminary findings, subject to confirmation in another study planned for the next lettuce production season.

Fusarium wilt of lettuce was first recognized in Arizona in 2001. Since this first discovery, the pathogen, Fusarium oxysporum f.sp. lactucae (Fol), has been recovered from infected lettuce plants from approximately 30 different fields. This fungus is a soil-borne pathogen that can remain viable in soil for many years. Cultural disease control measures, such as extended soil flooding and soil solarization, have shown promise in managing Fusarium wilt in other cropping systems. The specific objectives of this research were to repeat preliminary soil solarization and flooding experiments conducted last year and to evaluate the effect of preplant treatment of planting beds with either Vapam or soil solarization on the subsequent incidence of Fusarium wilt on lettuce. In a microplot study, soil naturally infested with Fol was flooded or solarized for 15, 30, 45 and 60 days, then bioassayed by transplanting and growing lettuce plants in samples of treated soil as well as nontreated soil. In field studies, plots were solarized for 40 days or treated with Vapam before planting to lettuce. In the microplot experiment, the severity of Fusarium wilt on lettuce grown in previously flooded or solarized soil was significantly less than that in nontreated soil. Additionally, there was no difference between flooding and solarization with respect to disease severity, as lettuce plants in both cases had virtually no symptoms of Fusarium wilt. Weight of the tops of lettuce plants was significantly greater for plants grown in flooded or solarized soil compared to that in nontreated soil. Furthermore, top growth in solarized soil was sometimes significantly greater than that in flooded soil. Compared to nontreated soil, root growth in solarized soil was significantly greater. In contrast, root growth in flooded soil was not significantly different than that recorded in nontreated soil. In the field studies, the incidence of lettuce plants with foliar symptoms of Fusarium wilt was reduced by an average of 42% when grown on solarized beds compared to nonsolarized beds. Preplant application of Vapam at rates of 30, 45 and 60 gallons of product per acre resulted in reductions in the incidence of Fusarium wilt of 38, 50, and 45%, respectively. Further work is needed to attempt to increase the reduction of disease recorded this past year. Refinements in our solarization technique as well as application methods for Vapam may increase the efficacy of these tools in reducing the incidence and severity of Fusarium wilt of lettuce.

"Grass pea (Lathyrus sativus) is an annual grain legume crop cultivated worldwide. Although considered underused, grass pea is an important source of protein for human and animal consumption in particular marginal areas. Furthermore, it is characterized by a remarkable resistance to abiotic stresses, such as drought, and biotic stress, such as particular pests and diseases. However, yield inconsistency due to sensitivity to specific diseases strongly limits its cultivation. One of these diseases is fusarium wilt, caused by the soil-borne fungi Fusarium oxysporum (Fo). Fusarium oxysporum promotes a devastating wilt(...)"
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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES
Common bean (Phaseolus vulgaris L.) is grown in Brazil in three different cropping seasons, and in diverse agroecosystems. In such different environments, the crop is exposed to several constraints responsible for yield losses, such as pathogenic organisms. Among common bean relevant diseases, fusarium wilt (Fusarium oxysporum f. sp. phaseoli), dry root-rot (Fusarium solani) and Curtobacterium wilt (Curtobacterium flaccumfaciens pv. flaccumfaciens) have similar symptoms, hindering diagnosis in the field, and whose identification in seed health testing is also limited. In both cases, identification at species level is an important step to manage this root pathogen complex, whose detection can be improved by molecular biology tools. Therefore, this study aimed to: 1) to develop and validate a multiplex PCR (m-PCR) method for simultaneous identification of three common bean pathogens, F. oxysporum f. sp. phaseoli, F. solani and C. flaccumfaciens pv. flaccumfaciens; and 2) develop an isothermal amplification of DNA (LAMP) method to detect of F. oxysporum f. sp. phaseoli on seeds. M-PCR method was developed for identification of isolated colonies, as well as infected seeds. In seeds, total DNA was obtained by alkaline lysis method, which inactivates nucleases during the extraction process. M-PCR allowed the identification of all pathogens, with detection of C. flaccumfaciens pv. flaccumfaciens, F. oxysporum f. sp. phaseoli and F. solani amplicons in agarose gel with respectively 306, 609 and 143 base pairs. Furthermore, m-PCR also reduced costs and time to detect Fusarium oxysporum f. sp. phaseoli from 10 days to three hours. It was not possible to develop an optimized protocol for detection of F. oxysporum f. sp. phaseoli by the LAMP method, using only the tf1 gene for design of primers, since such primers were functional only for amplifying large amounts of target DNA. Based on the negative results with LAMP, it is suggested that further studies should be performed using other DNA sequences available in GenBank database.
O feijoeiro-comum (Phaseolus vulgaris L.) é cultivado durante todo o ano no território brasileiro, em três épocas distintas e em vários agroecosistemas. Nestes ambientes distintos, a cultura está exposta a diversos fatores que causam perdas de rendimento, como o ataque de patógenos. Dentre as doenças do feijoeiro-comum encontram-se a murcha-de-fusarium (Fusarium oxysporum f. sp. phaseoli), a podridão-radicular-seca (Fusarium solani) e a murcha-de-curtobacterium (Curtobacterium flaccumfaciens pv. flaccumfaciens) que apresentam sintomas semelhantes, dificultando seu diagnóstico no campo, e cuja identificação em testes de sanidade de sementes também é limitada. Em ambos os casos, a identificação em nível de espécie é uma importante etapa do manejo deste complexo de patógenos, cuja detecção pode ser aperfeiçoada com a adoção de ferramentas de biologia molecular. Portanto, este estudo teve como objetivos: 1) Desenvolver e validar um método de multiplex PCR (m-PCR) para identificação simultânea de três espécies de patógenos do feijoeiro-comum, F. oxysporum f. sp. phaseoli, F. solani e C. flaccumfaciens pv. flaccumfaciens; e 2) desenvolver a técnica de amplificação isotérmica de DNA (LAMP) para detecção de F. oxysporum f. sp. phaseoli em sementes. O método de m-PCR foi desenvolvido para identificação de colônias isoladas bem como sementes infectadas. Nas sementes, o DNA total foi obtido pela lise alcalina, método que inativa nucleases durante o processo de extração. A m-PCR possibilitou a identificação de todos os patógenos, com detecção de C. flaccumfaciens pv. flaccumfaciens, F. oxysporum f. sp. phaseoli e F. solani em bandas formadas em gel de agarose respectivamente com 306, 609 e 143 pares de base. Além disso, a extração do DNA total das sementes pela lise alcalina em combinação com a m-PCR também possibilitou redução de custos e tempo de realização do diagnóstico de Fusarium oxysporum f. sp. phaseoli, de 10 dias para três horas. Não foi possível estabelecer um protocolo otimizado para detecção de F. oxysporum f. sp. phaseoli pelo método LAMP, utilizando somente o gene tf1 para desenho dos iniciadores, uma vez que, os iniciadores revelaram-se funcionais apenas para a amplificação com grandes quantidades de DNA alvo. Diante dos resultados obtidos com LAMP, sugere-se que estudos posteriores sejam realizados empregando outras sequências de DNA disponíveis no banco de dados GenBank.

Fusarium wilt of lettuce was first recognized in Arizona in 2001. The pathogen, Fusarium oxysporum f. sp. lactucae, has been recovered from infected lettuce plants in 27 different fields during the last three years. This fungus is a soil-borne pathogen that can remain viable in soil for many years. Historically, control of Fusarium wilt on crops other than lettuce, such as tomatoes and melons, has been achieved by planting cultivars resistant to the fungal pathogen. Large scale field trials were conducted during the 2002-03 and 2003-04 production seasons to evaluate existing lettuce cultivars for their relative susceptibility to Fusarium wilt. Among virtually all tested lettuce cultivars, the severity of disease in the first planting (early September) was much higher than that observed in the second planting (mid October), which in turn was higher than that observed in the third planting (early December). Soil temperatures differed considerably among plantings. In 2002-03 (or 2003-04) the average daily soil temperature at the 4-inch depth ranged from 65 to 85°F (70 to 94°F), 55 to 74°F (47 to 78°F), and 48 to 64°F (47 to 74°F) for the first, second and third plantings, respectively. In all three plantings, differences in disease severity were detected among the different types of lettuce, with head lettuce cultivars as a group being most susceptible and romaine cultivars collectively demonstrating the highest level of tolerance. Disease tolerance for specific cultivars was dependent on disease pressure. This is reflected in the comparative disease severity recorded in 2003-04 for specific cultivars (such as Beacon, Buccaneer, Coyote, Desert Heat, Lighthouse, Monolith, Red Tide, Sharpshooter, Sniper and Two Star) planted at each of the three different planting dates. Disease development began as early as the seedling stage and continued up to plant maturity, demonstrating the benefit of evaluating lettuce resistance in the field compared to greenhouse studies where plants are usually not carried to maturity before final disease ratings are performed. Data from these cultivar evaluation studies suggest that proper selection of planting date and cultivar would allow successful production of lettuce in fields infested with Fusarium oxysporum f. sp. lactucae.

This work encompasses studies on the phylogeny of F. oxysporum f. sp. lactucae, the development of a PCR-based seed assay for the detection of this fungus in seed, the potential of seed transmission of the fungus that may result in seed dissemination, and the genetic variation existing within pathogen populations. In phylogenetic analysis, the mtSSU and EF-1α sequences provided limited phylogenetic resolution and did not differentiate the lactucae isolates from other F. oxysporum isolates, while the IGS region resolved lactucae race 1 isolates as a monophyletic group with three other f. spp. of F. oxysporum. In all analyses, lactucae race 2 isolates comprised a separate lineage that was phylogenetically distinct. Based the IGS, PCR primers were designed for detection of the fungus, and a PCR-based seed assay was developed for detection of the fungus in seed. This assay allowed for detection of the pathogen from artificially infested seed lots with infestation rates as low as 0.5%. To investigate seedborne transmission, the moderately resistant cultivars Sharpshooter, Vulcan, and King Henry were inoculated and grown to maturity in the greenhouse. The pathogen was recovered from sections of surface disinfested inflorescence stalks at rates of 14.3 - 62.7% but not from the floral parts. The incidence of recovery from nondisinfested seeds was between 0.02% and 0.08%. The pathogen was not isolated from surface disinfested seeds suggesting that it was externally seedborne. The pathogen was recovered from pathogen-free seeds mixed with infested debris suggesting infested seed may contribute to recently documented dissemination of this pathogen worldwide. Isolates of Fusarium oxsyporum f. sp. lactucae were analyzed for genetic diversity using inter-simple sequence repeat molecular markers. Results revealed 2 main groups within the Arizona isolates corresponding to eight haplotypes in 2005, which evolved from 2 haplotypes in 2001. Haplotype 1-05 was widespread, occurring in two of the four countries where F. o. f. sp. lactucae has been reported. 23 haplotypes were identified among the California isolates that clustered into two subgroups. The clustering of isolates from Arizona suggests that there has been more than one introduction of the pathogen into Arizona.

Fusarium wilt of lettuce was first recognized in Arizona in 2001. Since this first discovery, the pathogen, Fusarium oxysporum f.sp. lactucae (Fol), has been recovered from infected lettuce plants from approximately 30 different fields. This fungus is a soil-borne pathogen that can remain viable in soil for many years. Cultural disease control measures, such as extended soil flooding and soil solarization, have shown promise in managing Fusarium wilt in other cropping systems. The specific research objective during the 2005 growing season was to further evaluate the effect of preplant solarization of planting beds on subsequent development of Fusarium wilt on lettuce. There was no significant difference between the short (28 days) and long (56 days) solarization period in the subsequent number of diseased lettuce plants; therefore, the disease incidence values for both solarization periods were combined and compared to nonsolarized plots. At each data collection date, the number of lettuce plants showing symptoms of Fusarium wilt was significantly lower in solarized beds compared to nonsolarized beds. At plant maturity (Nov 18), Fusarium wilt had claimed virtually all lettuce plants of the cultivar 'Lighthouse' growing in nonsolarized soil; however, only 19% of lettuce plants of the same cultivar growing in solarized soil showed disease symptoms. This equates to an 81% reduction in diseased plants in solarized soil compared to nonsolarized soil. The results of this field trial suggest that a 30-day summer solarization treatment of lettuce beds can significantly reduce the inoculum of Fusarium oxysporum f. sp. lactucae to levels that would allow substantial growth of a susceptible lettuce cultivar. Additional field studies are needed to refine the solarization process to potentially achieve further increases in efficiency of destroying propagules of Fusarium oxysporum f. sp. lactucae in infested fields.

Banana is the most important fruit crop in the world but ironically one of the crops least studied. This fruit constitutes a major staple food for millions of people in developing countries and also it is considered the highest selling fruit in the world market making this crop a very important export commodity for the producing countries. At the present time, one of the most significant constraints of banana production that causes significant economical losses are fungal diseases. Among these, Panama disease, also known as Fusarium wilt has been the most catastrophic. Panama disease is caused by the soil-borne fungus Fusarium oxysporum formae specialis (f.sp) cubense (FOC), which infects susceptible bananas through the roots causing a lethal vascular wilt. To date, the race 4 of this pathogen represents the most serious threat to banana production worldwide since most of the commercial cultivars are highly susceptible to this pathogen. Introduction of FOC resistance into commercial cultivars by conventional breeding has been difficult because edible bananas are sterile polyploids without seeds. Genetic transformation of banana, which has already been established in various laboratories around the world has the potential to solve this problem by transferring a FOC race 4 resistance gene into susceptible banana cultivars (eg. Cavendish cultivars). However, a FOC resistant (R) gene has not been isolated. Genes that confer resistance to Fusarium oxysporum have been isolated from tomato and melon using a map-based positional cloning approach. The tomato I2 and melon Fom-2 genes belong to the non-Toll/interleukin like receptors (TIR) subclass of nucleotide-binding site and leucine-rich repeat (NBS-LRR) R genes. These genes confer resistance only to certain races of F. oxysporum in their corresponding plant families limiting their use in other plant families. The fact that these two Fusarium resistance genes share the same basic non-TIR-NBS-LRR structure suggests a similar Fusarium resistance mechanism is shared between the families Solanaceae and Cucurbitaceae. This observation opens the possibility to find similar Fusarium resistance genes in other plant families including the Musaceae. A remarkable discovery of a population of the wild banana Musa acuminata subspecies (ssp.) malaccensis segregating for FOC race 4 resistance was made by Dr. Ivan Buddenhagen (University of California, Davis) in Southeast Asia. Research carried out at Queensland Department of Primary Industries (Australia) using this plant material has demonstrated that a single dominant gene is involved in FOC race 4 resistance (Dr. Mike Smith, unpublished results). Tissue-culture plantlets of this FOC race 4 segregating population were kindly provided to the Plant Biotechnology Program (Queensland University of Technology) by Dr. Mike Smith to be used in our research. This population holds the potential to assist in the isolation of a FOC race 4 resistance gene and other potential Fusarium resistance genes. The overall aims of this research were to isolate and characterise resistance gene candidates of the NBS-type from M. acuminata ssp. malaccensis and to identify and characterise potential Fusarium resistance genes using a combination of bioinformatics and gene expression analysis. Chapter 4 describes the isolation by degenerate PCR of five different classes of NBS sequences from banana (Musa acuminata ssp malaccensis) designated as resistance gene candidates (RGCs). Deduced amino acid sequences of the RGCs revealed the typical motifs present in the majority of known plant NBS-LRR resistance genes. Structural and phylogenetic analyses showed that the banana RGCs are related to non-TIR subclass of NBS sequences. The copy number of each class was estimated by Southern hybridisation and each RGC was found to be in low copy number. The expression of the RGCs was assessed by RT-PCR in leaf and root tissues of plants resistant or susceptible to Fusarium oxysporum f. sp. cubense (FOC) race 4. Four classes showed a constitutive expression profile whereas no expression was detected for one class in either tissue. Interestingly, a transcriptional polymorphism was found for RGC2 whose expression correlated with resistance to FOC race 4 suggesting a possible role of this gene in resistance to this devastating FOC race. Moreover, RGC2 along with RGC5 showed significant sequence similarity to the Fusarium resistance gene I2 from tomato and were chosen for further characterisation. The NBS sequences isolated in this study represent a valuable source of information that could be used to assist the cloning of functional R genes in banana. Chapter 5 describes the isolation and characterisation of the full open reading frame (ORF) of RGC2 and RGC5 cDNAs. The ORFs of these two banana RGCs were predicted to encode proteins that showed the typical structure of non-TIR-NBS-LRR resistance proteins. Homology searches using the entire ORF of RGC2 and RGC5 revealed significant sequence similarity to the Fusarium resistance gene I2 from tomato. Interestingly, the phylogenetic analysis showed that RGC2 and RGC5 were grouped within the same phylogenetic clade, along with the Fusarium resistance genes l2 and Fom-2. These findings suggest that the banana RGC2 and RGC5 are potential resistance gene candidates that could be associated with Fusarium resistance. The case of RGC2 is more remarkable because its expression was correlated to FOC race 4 resistance (Chapter 4). As a first step to test whether RGC2 has a role in FOC race 4 resistance, different expression constructs were made with the ORF of this sequence. One of the constructs contains a RGC2 putative promoter region that was successfully cloned in this work. These constructs will be used to transform susceptible banana plants that can then be challenged with FOC race 4 to assess whether resistance has been acquired by genetic complementation. The results of this thesis provide interesting insights about the structure, expression and phylogeny of two potential Fusarium resistance genes in banana, and provide a rational starting point for their functional characterisation. The information generated in this thesis may lead to the identification of a Fusarium resistance gene in banana in further studies and may also assist the cloning of Fusarium resistance genes in other plant species.

Banana is the most important fruit crop in the world but ironically one of the crops least studied. This fruit constitutes a major staple food for millions of people in developing countries and also it is considered the highest selling fruit in the world market making this crop a very important export commodity for the producing countries. At the present time, one of the most significant constraints of banana production that causes significant economical losses are fungal diseases. Among these, Panama disease, also known as Fusarium wilt has been the most catastrophic. Panama disease is caused by the soil-borne fungus Fusarium oxysporum formae specialis (f.sp) cubense (FOC), which infects susceptible bananas through the roots causing a lethal vascular wilt. To date, the race 4 of this pathogen represents the most serious threat to banana production worldwide since most of the commercial cultivars are highly susceptible to this pathogen. Introduction of FOC resistance into commercial cultivars by conventional breeding has been difficult because edible bananas are sterile polyploids without seeds. Genetic transformation of banana, which has already been established in various laboratories around the world has the potential to solve this problem by transferring a FOC race 4 resistance gene into susceptible banana cultivars (eg. Cavendish cultivars). However, a FOC resistant (R) gene has not been isolated. Genes that confer resistance to Fusarium oxysporum have been isolated from tomato and melon using a map-based positional cloning approach. The tomato I2 and melon Fom-2 genes belong to the non-Toll/interleukin like receptors (TIR) subclass of nucleotide-binding site and leucine-rich repeat (NBS-LRR) R genes. These genes confer resistance only to certain races of F. oxysporum in their corresponding plant families limiting their use in other plant families. The fact that these two Fusarium resistance genes share the same basic non-TIR-NBS-LRR structure suggests a similar Fusarium resistance mechanism is shared between the families Solanaceae and Cucurbitaceae. This observation opens the possibility to find similar Fusarium resistance genes in other plant families including the Musaceae. A remarkable discovery of a population of the wild banana Musa acuminata subspecies (ssp.) malaccensis segregating for FOC race 4 resistance was made by Dr. Ivan Buddenhagen (University of California, Davis) in Southeast Asia. Research carried out at Queensland Department of Primary Industries (Australia) using this plant material has demonstrated that a single dominant gene is involved in FOC race 4 resistance (Dr. Mike Smith, unpublished results). Tissue-culture plantlets of this FOC race 4 segregating population were kindly provided to the Plant Biotechnology Program (Queensland University of Technology) by Dr. Mike Smith to be used in our research. This population holds the potential to assist in the isolation of a FOC race 4 resistance gene and other potential Fusarium resistance genes. The overall aims of this research were to isolate and characterise resistance gene candidates of the NBS-type from M. acuminata ssp. malaccensis and to identify and characterise potential Fusarium resistance genes using a combination of bioinformatics and gene expression analysis. Chapter 4 describes the isolation by degenerate PCR of five different classes of NBS sequences from banana (Musa acuminata ssp malaccensis) designated as resistance gene candidates (RGCs). Deduced amino acid sequences of the RGCs revealed the typical motifs present in the majority of known plant NBS-LRR resistance genes. Structural and phylogenetic analyses showed that the banana RGCs are related to non-TIR subclass of NBS sequences. The copy number of each class was estimated by Southern hybridisation and each RGC was found to be in low copy number. The expression of the RGCs was assessed by RT-PCR in leaf and root tissues of plants resistant or susceptible to Fusarium oxysporum f. sp. cubense (FOC) race 4. Four classes showed a constitutive expression profile whereas no expression was detected for one class in either tissue. Interestingly, a transcriptional polymorphism was found for RGC2 whose expression correlated with resistance to FOC race 4 suggesting a possible role of this gene in resistance to this devastating FOC race. Moreover, RGC2 along with RGC5 showed significant sequence similarity to the Fusarium resistance gene I2 from tomato and were chosen for further characterisation. The NBS sequences isolated in this study represent a valuable source of information that could be used to assist the cloning of functional R genes in banana. Chapter 5 describes the isolation and characterisation of the full open reading frame (ORF) of RGC2 and RGC5 cDNAs. The ORFs of these two banana RGCs were predicted to encode proteins that showed the typical structure of non-TIR-NBS-LRR resistance proteins. Homology searches using the entire ORF of RGC2 and RGC5 revealed significant sequence similarity to the Fusarium resistance gene I2 from tomato. Interestingly, the phylogenetic analysis showed that RGC2 and RGC5 were grouped within the same phylogenetic clade, along with the Fusarium resistance genes l2 and Fom-2. These findings suggest that the banana RGC2 and RGC5 are potential resistance gene candidates that could be associated with Fusarium resistance. The case of RGC2 is more remarkable because its expression was correlated to FOC race 4 resistance (Chapter 4). As a first step to test whether RGC2 has a role in FOC race 4 resistance, different expression constructs were made with the ORF of this sequence. One of the constructs contains a RGC2 putative promoter region that was successfully cloned in this work. These constructs will be used to transform susceptible banana plants that can then be challenged with FOC race 4 to assess whether resistance has been acquired by genetic complementation. The results of this thesis provide interesting insights about the structure, expression and phylogeny of two potential Fusarium resistance genes in banana, and provide a rational starting point for their functional characterisation. The information generated in this thesis may lead to the identification of a Fusarium resistance gene in banana in further studies and may also assist the cloning of Fusarium resistance genes in other plant species.

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A indução de supressividade a Fusarium oxysporum f. sp. cubense (Foc) em bananeira por meio de produtos orgânicos foi avaliada pela incorporação ao solo, artificialmente infestado com o patógeno, de biocarvão de serragem de Eucalyptus (BC), casca de camarão (CC), composto de lodo de esgoto (LE), concha de marisco (CM), bio-óleo de casca de arroz (BA) e de serragem de Eucalyptus (BE), emulsão (EP) e hidrolisado de peixe (HP). Características químicas do solo; área abaixo da curva de progresso do índice da doença (AACPID); severidade final; descoloração interna do rizoma; respiração microbiana por meio da liberação de CO2; comunidade de F. oxysporum, fungos e bactérias totais; a inibição do crescimento micelial e da germinação de microconídios pelos extratos aquosos e pela liberação de compostos voláteis tóxicos foram os parâmetros avaliados. A CC e o LE reduziram a severidade externa (50 e 37%, respectivamente) e a descoloração interna do rizoma (56 e 36%, respectivamente). As CC e CM reduziram o desenvolvimento das plantas nas fases iniciais de cultivo, mostrando sintomas de fitotoxicidade. A elevação do pH, condutividade elétrica (CE), K, Mg, S, Na, Fe, Zn, CO2 e comunidade de bactérias foram as características que apresentaram maior influência na redução da severidade, enquanto que o aumento no pH, CE, CO2, P, Ca, S, Na e B também contribuíram para reduzir a população de F. oxysporum no solo. O BA, o BE e o HP reduziram em 94, 84 e 40% a AACPID, respectivamente. Os teores de Zn, a inibição do crescimento micelial e inibição da germinação dos microconídios pela ação de compostos voláteis e de extratos aquosos foram os mecanismos que mais explicaram o controle ...
The induction of suppressiveness to Fusarium wilt in banana by the incorporation of Eucalyptus sawdust biochar (BC), shrimp shells (CC), composted sewage sludge (LE), clamshell (CM), bio-oil of rice husk (BA) and bio-oil of Eucalyptus sawdust (BE), fish emulsion (EP) and fish hydrolyzate (HP) to soil was evaluated. Soil chemical characteristics; the area under the curve of progress of the disease index (AUPCDI), final severity and internal discoloration of the rhizome; microbial respiration through the release of CO2; the populations of F. oxysporum, fungi and total bacteria; and inhibition of mycelial growth and microconidia germination through the compounds volatile toxic and aqueous extracts were the parameters evaluated. The CC and LE reduced final severity (50 and 37%, respectively) and the internal discoloration of the rhizome (56 and 36%, respectively). The incorporation of CC and CM reduced plant development in the early stages of cultivation, showing symptoms of phytotoxicity. The increase in pH, electric ...

Fusarium wilt of lettuce was first recognized in Arizona in 2001. Since this initial discovery, the pathogen, Fusarium oxysporum f.sp. lactucae (Fol), has been recovered from infected lettuce plants from over 40 different fields. This fungus is a soil-borne pathogen that can remain viable in soil for many years. Soil solarization has shown promise in managing Fusarium wilt in other cropping systems as well as in lettuce field trials conducted during 2004 and 2005. In an effort to maximize the solarization effect on subsequent disease development, the following factors were evaluated in a 2006 field trial: 1) solarization of unshaped versus preshaped beds, 2) the effect of soil moisture on solarization efficiency of preshaped beds, and 3) effect of lettuce type on Fusarium wilt incidence after solarization. The entire field was flood irrigated on Jun 21. Plots were solarized during Jul and/or Aug by covering beds with 1-mil thick clear plastic. During the solarization treatment from Jul 3 to Sep 10, the mean soil temperature in preshaped solarized beds at a depth of 2 and 9 inches was 116 and 95°F, respectively, and 102 and 97°F, respectively, in beds not covered with plastic. When solarization was initiated 15 days after soil irrigation, a 20% reduction in Fusarium wilt was recorded for a crisphead lettuce cultivar grown on solarized unshaped beds compared to a 56% reduction in disease when the same crisphead cultivar was grown on preshaped solarized beds. There was no significant difference between a one and two month solarization period in the subsequent number of diseased lettuce plants. Solarization of preshaped beds 15 days after irrigation for one month reduced Fusarium wilt on crisphead lettuce by 56%, whereas the same solarization period initiated seven days after irrigation resulted in a 96% reduction of disease. The same one-month solarization period started one week after soil irrigation reduced the incidence of Fusarium wilt on green leaf (Two Star) and romaine (Green Towers) by 97 and 88%, respectively, compared to plants grown on unsolarized beds. The data show that summer solarization of moist soil can 1) destroy propagules of Fusarium oxysporum f. sp. lactucae in infested fields and 2) be a useful cultural management tool to significantly reduce the incidence of Fusarium wilt in a subsequent crop of lettuce.

Orientador: Wagner Bettiol
Coorientador: Carlos Gilberto Raetano
Banca: Antonio Carlos Maringoni
Banca: Miguel Angel Dita Rodriguez
Resumo: A indução de supressividade a Fusarium oxysporum f. sp. cubense (Foc) em bananeira por meio de produtos orgânicos foi avaliada pela incorporação ao solo, artificialmente infestado com o patógeno, de biocarvão de serragem de Eucalyptus (BC), casca de camarão (CC), composto de lodo de esgoto (LE), concha de marisco (CM), bio-óleo de casca de arroz (BA) e de serragem de Eucalyptus (BE), emulsão (EP) e hidrolisado de peixe (HP). Características químicas do solo; área abaixo da curva de progresso do índice da doença (AACPID); severidade final; descoloração interna do rizoma; respiração microbiana por meio da liberação de CO2; comunidade de F. oxysporum, fungos e bactérias totais; a inibição do crescimento micelial e da germinação de microconídios pelos extratos aquosos e pela liberação de compostos voláteis tóxicos foram os parâmetros avaliados. A CC e o LE reduziram a severidade externa (50 e 37%, respectivamente) e a descoloração interna do rizoma (56 e 36%, respectivamente). As CC e CM reduziram o desenvolvimento das plantas nas fases iniciais de cultivo, mostrando sintomas de fitotoxicidade. A elevação do pH, condutividade elétrica (CE), K, Mg, S, Na, Fe, Zn, CO2 e comunidade de bactérias foram as características que apresentaram maior influência na redução da severidade, enquanto que o aumento no pH, CE, CO2, P, Ca, S, Na e B também contribuíram para reduzir a população de F. oxysporum no solo. O BA, o BE e o HP reduziram em 94, 84 e 40% a AACPID, respectivamente. Os teores de Zn, a inibição do crescimento micelial e inibição da germinação dos microconídios pela ação de compostos voláteis e de extratos aquosos foram os mecanismos que mais explicaram o controle ...
Abstract: The induction of suppressiveness to Fusarium wilt in banana by the incorporation of Eucalyptus sawdust biochar (BC), shrimp shells (CC), composted sewage sludge (LE), clamshell (CM), bio-oil of rice husk (BA) and bio-oil of Eucalyptus sawdust (BE), fish emulsion (EP) and fish hydrolyzate (HP) to soil was evaluated. Soil chemical characteristics; the area under the curve of progress of the disease index (AUPCDI), final severity and internal discoloration of the rhizome; microbial respiration through the release of CO2; the populations of F. oxysporum, fungi and total bacteria; and inhibition of mycelial growth and microconidia germination through the compounds volatile toxic and aqueous extracts were the parameters evaluated. The CC and LE reduced final severity (50 and 37%, respectively) and the internal discoloration of the rhizome (56 and 36%, respectively). The incorporation of CC and CM reduced plant development in the early stages of cultivation, showing symptoms of phytotoxicity. The increase in pH, electric ...
Mestre

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Conselho Nacional de Desenvolvimento Científico e Tecnológico
The fusarium wilt caused by Fusarium oxysporum f. sp. lycopersici (Fol), is one of the major problems in tomato cultivation especially in green house crop. The soil infestation with this pathogen can make the green house cultivation unviable, therefore periodic fumigation is recommended to maintain low inoculum level in soil. This study was done to evaluate the fumigant effect of the mustard essential oil (MEO), containing 90% allyl isothiocyanate, to control Fol. In vitro bioassays were done to determine its effect on mycelial growth, sporulation and germination of conidia and clamydospores, with use of a wild Fol and benomyl resistant mutant (Folm). The fungal cultures in Petri plates were fumigated with different concentration of the MEO for 24 or 48 h, and then incubated in MEO free atmosphere. For all fungal propagules, the estimated DE50 was lowest if the fumigation was done for 48 h. The mycelium and conidia of the Fol were more susceptible to MEO than chlamydospores. The MEO did not affect sporulation. Fumigation with MEO was also evaluated for eradication of the chlamydospores of Folm in soil. Initially, the interaction between dose (0, 50, 100 or 150μL/L) and exposure time was determined (2, 4, 6 or 8 days). The soil infested with 2000 ±200 chlamydospores/g was placed in flasks, and after adding the requited amount of MEO the flasks were hermetically sealed. After each exposure period, the inoculum density of the fungus was determined by plating the soil dilutions on benomyl enriched galactosenitrate agar. The regression equation revealed that at dose of 125μL/L an exposure period of 5.4 days was required to eradicate Folm. To determine the fumigant effect of MEO in the green house, 20L of soil infested with 4000 ±250 chlamydospores/g was placed in the plastic bags of 30L, and treated with 0, 50, 100 or 150μL/L of MEO. The bags were then sealed and stored. After 7-days exposure period, the soil was distributed into 4L-plastic pots, and one 20-day old tomato seedling was transplanted into each pot. At 15-day interval, soil from each pot was sampled at 15-day interval to follow the population dynamic of the fungus. The disease progress was accompanied by leaf chlorophyll analysis leaves, and the final severity was evaluated by use of a numerical at the end of 60 days. It was found that the soil fumigation with 150μL/L of MEO reduced the Folm inoculum density by 95% and the disease severity was less than 15%.
A murcha de fusário, causada por Fusarium oxysporum f. sp. lycopersici (Fol), é um problema comum em campos de produção de tomate, especialmente quando o cultivo é realizado em ambiente protegido. Solos infestados por este patógeno podem inviabilizar a produção em estufas, sendo recomendada a fumigação periódica, visando à manutenção de um baixo nível de inóculo no solo. Este trabalho teve como objetivo avaliar o efeito fumigante do óleo essencial de mostarda, que é composto por 90% de isotiocianato de alila (ITCA), na redução de inóculo e no controle da murcha vascular causada por Fol. Foram realizados bioensaios in vitro de crescimento micelial, formação de conídios e germinação de conídios e de clamidósporos. Para os testes, foram utilizados um isolado selvagem (Fols) e um mutante resistente ao benomil (Folm), os quais foram fumigados com ITCA, em diferentes doses, dentro de recipientes plásticos vedados, por períodos de 24 ou 48 horas. Após a fumigação, as placas contendo as culturas foram incubadas na ausência dos vapores do produto até a avaliação. Os menores valores de DE50 foram estimados para o período de 48 horas de exposição, tanto para o bioensaio de crescimento micelial como para os de germinação de conídios e de clamidósporos. Verificou-se que os conídios foram os propágulos de Fol mais sensíveis ao produto e os clamidósporos os mais resistentes. O ITCA não afetou significativamente a formação de conídios pelos isolados. Avaliou-se também a eficiência do produto na erradicação de clamidósporos de Folm no solo. Inicialmente, foi estudada a interação entre doses (0, 50, 100 e 150μL/L) e tempo de exposição (2, 4, 6 e 8 dias) ao ITCA. Solo infestado com 2000 ±200 clamidósporos/g foi transferido para erlenmeyers, que receberam a dose desejada, sendo, em seguida, hermeticamente vedados. Após exposição, a população do fungo foi determinada por meio de plaqueamento de diluições em série em meio seletivo para F. oxysporum acrescido de benomil. A partir da equação de regressão gerada, pôde-se estimar que seria necessária uma fumigação de solo com 125μL/L por períodos superiores a 5,4 dias para erradicação de Folm no solo. Para determinar o efeito de ITCA em casa de vegetação, 20L de solo infestado com 4000 ±250 clamidósporos/g foram colocados em sacos de polietileno de 30L, os quais receberam as doses de 0, 50, 100 ou 150μL/L sendo, posteriormente, vedados, permitindo a fumigação por 7 dias. Decorrido este período, o solo foi transferido para vasos de 4L, os quais receberam uma muda de tomate com 20 dias de idade. As plantas foram cultivadas por 60 dias, sendo retiradas amostras quinzenais de solo para acompanhamento da dinâmica populacional do fungo no solo. Através de análise do conteúdo de clorofila nas folhas, acompanhou-se o desenvolvimento da doença e a severidade final foi avaliada por meio de escala de notas. Foi verificado que a fumigação com 150μL/L de ITCA reduziu em mais de 95% a população de Folm no solo e que a severidade da doença aos 60 dias foi inferior a 15%.

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Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq
The Fusarium wilt caused by Fusarium oxysporum f. sp. lycopersici, is an important disease of tomato (Solanum lycopersicon L.) in Northeastern Brazil. Order to select genotypes with potential for use in the disease management, 60 strains were evaluated (F7 generation) from the crossing of access BHRS-2, 3 and Viradoro cultivar in relation to isolates from the physiologic races 2 and 3 of F. oxysporum f. sp. lycopersici. Seedlings 21 days old were inoculated using the method of cutting the roots and soak in the suspension of conidia of the pathogen. The evaluation was performed after 21 days, with the scale of grades ranging from 1 to 5. The genotypes were grouped into five classes of reaction. Most genotypes (73.3%) behaved as highly resistant to the race 2 isolate, while 45.0% were classified as susceptible and 28.3% as highly susceptible to the race 3 isolate. Only the L-1 strain showed high resistance reaction to both isolates. The stability of this line of resistance was evaluated on five isolates ofeach race (2 and 3). The line L-1 showed high levels of resistance to all race 2 isolates, therefore indicating high stability of resistance. However, for race 3 isolates, this strain showed three distinct classes of reaction, ranging from highly resistant to susceptible, indicating instability of resistance to this race.
A murcha-de-fusário, causada por Fusarium oxysporum f. sp. lycopersici, é uma importante doença do tomateiro (Solanum lycopersicon L.) no Nordeste brasileiro. Visando selecionar genótipos com potencial de utilização no manejo da doença, foram avaliadas 60 linhagens (geração F7) oriundas do cruzamento entre o acesso BHRS-2,3 e a cultivar Viradoro, em relação a isolados das raças fisiológicas 2 e 3 de F. oxysporum f. sp. lycopersici. Mudas com 21 dias de idade foram inoculadas pelo método do corte de raízes e imersão na suspensão de conídios do patógeno. A avaliação foi realizada após 21 dias, com o auxílio de escala de notas de 1 a 5, para agrupamento dos genótipos em cinco classes de reação. A maioria dos genótipos (73,3%) se comportou como altamente resistente ao isolado da raça 2, enquanto 45,0% foram classificados como suscetíveis e 28,3% como altamente suscetíveis ao isolado da raça 3. Somente a linhagem L-1 apresentou reação de alta resistência aos dois isolados de ambas as raças. A estabilidade da resistência dessa linhagem foi avaliada em relação a cinco isolados de cada raça (2 e 3) do patógeno. A linhagem L-1 apresentou reação de alta resistência a todos os isolados da raça 2, evidenciado estabilidade da resistência. No entanto, em relação aosisolados da raça 3, essa linhagem apresentou três classes de reação distintas, variando de altamente resistente a suscetível, indicando instabilidade da resistência à essa raça.

Management of Fusarium wilt of banana, one of the most important diseases of agricultural crops, is complicated and involves the consideration of factors such as the biology, epidemiology and population structure of the pathogen, and genetic resources and production practices of the crop. The development of an integrated disease management programme, therefore, is of great importance in countries where the Fusarium wilt pathogen, Fusarium oxysporum f.sp. cubense Foc, has been introduced into banaria fields, and where resistant cultivars are not acceptable to local markets. To achieve this, it is important to investigate new management strategies and to review methods that have been less successful in the past. These management practices need to be practical and affordable. Since certain cultural practices have proven to be effective, management practices that could compliment them should be considered. This thesis has attempted to investigate such practices in order to develop an integrated disease management programme for Fusarium wilt of banana. One of the most important findings of this study, was that the surface sterilant previously used to prevent the introduction of the Fusarium wilt into uninfected areas in South Africa, are not effective. The sterilants Sporekill and Prazin proved to be highly effective, and are now recommended to replace the sterilants previously used. Several fungicides reduced mycelial growth of Foc in vitro, with the OMI fungicides and Benomyl found to be the most effective. The same fungicides reduced the disease severity of Fusarium wilt in the greenhouse significantly, especially when they were applied as root dip treatments. None of the fungicides found effective against Foc have been evaluated in the field against Foc before. The next step, therefore, would be to evaluate root dip treatments combined with drench treatment in the field. Although it is expected that these fungicides might have a negative effect on the microbial populations in the soil, this has yet to be investigated. Fungicides may even weaken or stress the pathogen, making it more vulnerable for the action of an effective biocontrol agent or agents. Chemical activators are probably one of the most attractive strategies to combat Fusarium wilt of banana, since it stimulate the plants' own defence system. Banana plantlets were found to be quite sensitive to the amount and method whereby chemical activators were applied. The activator benzo-(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester induced resistance against Foc on the susceptible Williams cultivar in the greenhouse, but not in the field. In field studies, environmental conditions were much more variable than in the greenhouse, which made it difficult to evaluate the effectiveness of chemical activators. Sodium nitroprusside and a product containing the harpin protein showed promising results on the Williams and DRSI cultivars, respectively. These activators need to be considered as part of an integrated disease management programme. Since they are not directly applied to the soil, they will not have a negative effect on the microbial populations in the soil. Several Fusarium isolates had been collected from banana fields with disease suppressive soils in Kiepersol, South Africa. Most of these isolates were F. oxysporum, and with the exception of one isolate, proved to be non-pathogenic to banana plants. A PCR-based restriction fragment length polymorphism (RFLP) analysis of the intergenic spacer region of the ribosomal RNA operon grouped the non-pathogenic F. oxysporum isolates into 12 distinct genotypes. A great diversity could be seen among the non-pathogenic isolates compared to the pathogenic Foc isolates. The known-biological control agent F047 grouped with three of the South African isolates, while the one pathogenic isolate grouped with the pathogenic Foc from diseased Cavendish bananas in South Africa By using PCR-RFLPs, we were able to rapidly characterize the structure of non-pathogenic isolates of F. oxysporum in disease suppressive soils in Kiepersol. This could assist us in our search for potential biological control agents for Fusarium wilt of banana. Representative isolates from the 12 genotype groups were selected for evaluation of Fusarium wilt suppressive properties in banana. These non-pathogenic F. oxysporum isolates appeared to be good biological control candidates and was compared to known biological control agents and commercial biological control products. Fourteen of the non-pathogenic isolates, the combination of two Trichoderma strains form suppressive soils in South Africa, and two Pseudomonas fluorescens isolates were found to significantly reduce Fusarium wilt development in the greenhouse. The commercial products Patostop, B-rus and a mixture of arbuscular mycorrhizae were also found to suppress the disease severity of Foc significantly. The well-know biological control agent F047 proved to be not effective. Results concluded that two of the non-pathogenic F. oxysporum isolates and the two P. fluorescens isolates, one of which was the well-known WCS 417, were the most effective of all the agents evaluated. Since combinations of biocontrol agents may provide even more consistent and effective control than a single agent, future research will include the combination of biocontrol agents found effective in this study. It would also be of great value to determine the mode of action of these isolates, so that isolates with different modes of action could be combined to enhance the suppression effect. Biological control can be a very useful component of an integrated disease management programme, since the effective agent or agents can easily be established on tissue culture banana plantlets before they are planted in the field. AFRIKAANS : Een van seker die mees belangrikste grondgedraagte siektes in lanbou, is Fusarium verwelksiekt van piesangs. In Suid-Afrika, is die siekte verantwoordelik vir emstige verliese in die piesang produksie. Aangesien daar geen weerstandbiedende kultivars beskikbaar is wat deur die mark aanvaar word nie, is dit van kardinale belang dat 'n geintegreerde siekte beheer program vir Suid-Afrika ontwikkel word. Voordat so 'n program saamgestel kan word, is dit belangrik dat verskeie faktore aangaande die patogeen en piesang poduksiepraktyke in ag geneem moet word. Beheermaatreëls moet prakties en bekostigbaar wees, en moet die reeds bestaande praktyke kan bevoordeel. Studies wat in hierdie tesis aangebied word, oorweeg beheermaatreëls wat gekombineer kan word met die huidige praktyke, nadat vorige praktyke ook in ag geneem is. Daar word gesoek na nuwe meer doeltreffende en ekonomiese metodes om siektes te beheer. Metodes wat doeltreffend aangewend kan word om die voorkoms van die siekte te vermirider. In vitro en in vivo studies het getoon dat die DMI swamdoders en Benomil die groei van die patogeen en die ontwikkeling van Fusarium verwelksiekte die meeste onderdruk. Die beste resultate is in die glashuis gevind nadat die wortels van plante in die middels geweek is. Positiewe resultate is ook verkry met die grondtoediening van Benomil 'n week nadat plante geplant is in Foc geïnfekteerde grond. Die chemiese beheer van Fusarium verwelsiekte kan verder ondersoek word deur die effek van die grondtoedienings en wortelbehandelings in die veld te ondersoek. Daar word egter verwag dat die swamdoders moontlik 'n negatiewe uitwerking op die mikrobiese aktiwiteit in die grond kan veroorsaak. Die gebruik van effektiewe ontsmettingmiddels is uiters belangrik vir die voorkomende beheer van Fusarium verwelkdiekte op piesangs. Die ontsmettingmiddel, koper oxichloried, wat tot onlangs in Suid Afrika gebruik was, is ondoeltreffend gevind vir ontsmettingsdoeleindes. Prazin en Sporekill, twee omgewingsvriendelike middels, is baie effektief gevind en word dus aanbeveel vir die ontsetting van voertuie, skoene en veld toerusing. Chemiese plant aktiveerders stimuleer plante om hulleself te beskerm deur middel van weerstandsmeganisms. Piesang plante het sensitiwiteit getoon toonoor die konsentrasie en die toedieningsmetode van hierdie chemiese aktiveerders. In die glashuisproewe het die aktiveerder benzo-(1,2,3)thiadiazole-7-carbothioic suur S-metiel ester weerstand gestimuleer in die Williams kultivar. As gevolg van veranderende toestande in die veld was dit moeiliker om die chemiese aktiveerders se werking te evalueer. Nogtans het die middels natrium nitroprussied en 'n produk wat die protein harpin bevat die voorkoms van siekte op die Williams en DRS 1 plante verlaag. Chemiese aktiveerders behoort sterk oorweeg te word as deel van 'n geintegreerde beheer program, aangesien chemiese aktiveerders nie direk tot die grond aangewend word nie, en geen negatiewe uitwerking op die natuurlike mikrobiese populasies in die grond uitoefen nie. Verskeie Fusarium isolate is geisoleer vanuit siekte onderdukkende gronde in die Kiepersol area van Suid-Afrika. Die meeste van die isolate is geidentifiseer as F. oxsysporum. 'n PKR-gebaseerde restriksie fragment lengte polimorfisme (RFLP) ontleding van die "intergenic spacer region" van die ribosomale DNS operon het die niepatogeniese F. oxysporum isolate in 12 verskillende genotypes opgedeel. 'n Groot diversiteit was sigbaar onder die nie-patogeniese isolate in vergelyking met die patogeniese foc isolate. Die bekende beheer agent, Fo47 het gegroepeer saam met drie van die Suid Afrikaanse nie-patogene. Hierdie tegniek het ons in staat gestel om die nie-patogeniese populasie van onderdrukkende gronde in Kiepersol vinnig te karakteriseer en potentiele biologiese agente te identifiseer. Verteenwoordigende isolate van die 12 genotipiese groepe wat geidnetifiseer is, is geselekteer vir verdere evaluasie. Dit is gevind dat die isolate goeie kandidate vir moontlike bio-beheer agente maak. Die onderdrukkingsvermoe van die nie-patogene is vergelyk met die van bekende bio-beheer agente en komersiele produkte wat beskikbaar is. Veertien van die nie-patogene, die kombinasie van twee Trichoderma spp., en twee Pseudomonas fluorescens isolate het die siekte ontwikkeling van Fusarium verwelking merkwaardig onderdruk in die glashuis. Die komersiele produkte Patostop®, B-rus en die kombinasie van twee mycorrhizae isolate is ook gevind om die voorkoms van siekte te verlaag. Die wel-bekende biobeheer agent Fo47 is oneffektief gevind teen Fusarium verwelksiekte van piesangs. Resultate van die studie het bewys dat twee van die nie-patogeniese F.Oxysporum isolate en twee P. Fluorescens isolate, waarvan een die welbekende WCS 417 is, uiters effektiewe beheer agente teen Foc is. Toekomstige studies sal fokus op die kombinasie van die bio-beheer agente wat die meeste potensiaal getoon het in die studie, asook hulle meganismes van werking. Biologiese beheer is van groot waarde vir 'n geïntegreerde beheer program. Dit kan maklik met bestaande beheer maatreëls gekombineer word en potensiële biologiese beheer agente kan vooraf op weefselkultuur plante in die kwekery gevestig word.
Dissertation (MSc)--University of Pretoria, 2011.
Microbiology and Plant Pathology
unrestricted

碩士
國立中興大學
生命科學系所
104
Banana is one of the most important crops in the world, and has wealth of nutritional value. The production of the banana is seriously affected by the Fusarium oxysporum f. sp. cubense tropical race 4. If we can inhibit Fusarium wilt of the banana, then it will increase the annual production of banana and thus reducing the use of chemicals. Endophytes may not only inhibit Fusarium wilt but also promote the growth of the bananas by inoculating Cavendish banana seedlings. In this experiment, two plant endophytes Burkholderia cenocepacia 869T2 and Bacillus amyloliquefaciens BPRB-37 at different concentrations were inoculated in the“Pei Chiao”Cavendish banana seedlings. In greenhouse experiment, the heights of 869T2-inoculated“Pei Chiao”as 6% taller than those without after seven months. In field experiment, the heights of 869T2 and BPRB-37-inoculated“Pei Chiao”plants as 33% taller than those without inoculation after seven months. In greenhouse experiment, all“Pei Chiao”plants showed no pathogenesis after seven months. In field experiment, 869T2-inoculated“Pei Chiao”plants morbidity was 20%, and those without 869T2 inoculation treatments were 40%, indicating that 869T2 treatment could decrease morbidity of“Pei Chiao”plants affected by Foc TR4 50% after seven months. In order to elabrate the plant growth promotion (PGP) gene, the next generation sequencing maked a technology was applied . We predict 869T2 could produce substances to promote the growth of the plant, and has antifungal function,while another one BPRB-37 just has antifungal function.

Fusarium oxysporum f.sp. cubense Schlecht (Foc), causal agent of Fusarium wilt of banana (Panama disease), is considered to be one of the most serious threats to banana production in the world. There is no effective control measure for Fusarium wilt, except for the replacement of susceptible with resistant banana varieties. However, resistant varieties are not always acceptable to producers and local consumer markets. A greater awareness of the detrimental effect of chemicals on the environment has stimulated research on biological control of plant pathogens. The use of indigenous microorganims, such as non-pathogenic F. oxysporum and the bacterium Pseudomonas fluorescens, therefore, offers not only an environmentally safe but also an economical approach to combat Fusarium wilt of banana as part of an integrated disease management strategy. Non-pathogenic F. oxysporum and P. fluorescens isolates have previously been isolated from the root rhizosphere in disease suppressive soils. These isolates have the ability to reduce the incidence of Fusarium wilt in greenhouse pathogenicity trials. In this study we had hoped to expand on existing knowledge on the biological control of Fusarium wilt of banana with non-pathogenic endophytic F. oxysporum and P. fluorescens. Isolates that significantly suppress disease development in greenhouse trials were tested under field conditions. Physiological and histological studies were also performed to understand the modes of action of putative biological control agents. For the histological investigations, non-pathogenic F. oxysporum isolates were modified with green and red fluorescent proteins. Chapter 1 depicts a general overview of the biological control of Fusarium wilt diseases of agricultural crops. This chapter addresses the biology and pathogenesis of F. oxysporum, before strategies to control Fusarium wilt are discussed. The application of biological control organisms was analysed in terms of potentially useful organisms, where they can be isolated, and their possible modes of action. Finally, factors that influence biological control of Fusarium wilt diseases are discussed. A good source of prospective biocontrol agents is suppressive soils. In Chapter 2, non-pathogenic F. oxysporum isolates were collected from healthy banana roots in disease suppressive soil. Random Fragment Length Polymorphisms of the intergenic spacer region were then applied to group the non-pathogenic F. oxysporum isolates into genotypes, from which candidates were selected for biological control studies. The selected endophytes were then inoculated onto banana roots to determine their ability to act as biocontrol agents against Foc. The isolates that protected banana best against Fusarium wilt in the greenhouse, together with P. fluorescens WCS 417, were tested in the field to determine whether these isolates could effectively reduce disease incidence in an uncontrolled environment. The ability of non-pathogenic F. oxysporum and P. fluorescens WCS 417 to induce systemic resistance in Cavendish banana plants against Foc was investigated in Chapter 3 with the use of a split-root technique. The putative biocontrol agents were inoculated, separately and in combination, on one half of the roots in a split-root experiment, while the other half was challenged by a pathogenic isolate of Foc. Five different phenolic acids were assayed which included total soluble phenolic acids, non-conjugated (free acids) phenolic acids, ester-bound phenolic acids, glycosidebound phenolic acids and cell wall-bound phenolic acids. The knowledge gained will contribute to the understanding of how the biocontrol agents may induce defense responses in banana roots against Foc. Non-pathogenic isolates of F. oxysporum were transformed with the green fluorescent protein (GFP) and DsRed-Express genes in Chapter 4. These isolates were used to visualise their interactions with a GFP-transformed Foc isolate on the banana root in a non-destructive manner by means of confocal laser scanning microscopy (CLSM) in Chapter 5. The ability of non-pathogenic F. oxysporum and P. fluorescens WCS 417 to induce structural changes was also investigated with a split-root system using the CLSM. Antibioses as a mode of action of the two potential biocontrol agents was tested in vitro. Understanding the modes of action of non-pathogenic F. oxysporum and P. fluorescens WCS 417 are important when considering strategies for the implementation of these isolates in an integrated disease management strategy against Fusarium wilt of banana.
Dissertation (MSc (Plant Pathology))--University of Pretoria, 2011.
Microbiology and Plant Pathology
unrestricted